In a cell culture apparatus in which microorganisms, animal or plant cells, or the like are cultured, a state of cultured cells is monitored so as to efficiently culture the target cells while controlling flow stirring or gas vent in a culture vessel or supply of a liquid medium. Other items to be monitored include a culture temperature, a pH, a dissolved oxygen concentration, a dissolved carbon dioxide concentration, a cell concentration, a medium component such as amino acid including glucose and glutamine, a metabolic component of the cell such as lactic acid and amino acid, and a cellular product such as protein.
If the culture vessel used is made of stainless steel or glass, an electrode type measurement sensor suited for the culture temperature, pH, dissolved oxygen concentration, and dissolved carbon dioxide concentration of the monitoring items described above is installed on a roof in an upper part of the culture vessel or on a wall surface thereof, so as to monitor the culture condition.
The medium component, the metabolic component, the cellular product, or the like is measured by sampling part of the cell culture solution in the culture vessel under a bacteria-free condition and measuring the target item using an appropriate measurement unit.
The cell concentration is typically calculated by counting the number of cells using a sampled cell culture solution or by measuring light turbidity. Similarly to the above-described electrode type measurement sensor, a probe type turbidity sensor which is used by being inserted in a culture vessel is currently in practical use and can also be used for monitoring the cell concentration.
When the electrode type measurement sensor is used, before starting a culture of cells, it is necessary for the electrode type measurement sensor to be subjected to sterilization in an autoclave in a state where the sensor is set in a culture vessel, or by circulating steam in a stationary culture vessel. When the cell culture solution is sampled, it is necessary for extract piping of the cell culture solution to be subjected to sterilization using steam, flame, alcohol, or the like each time.
Given these circumstances, in order to quickly set up a cell culture apparatus, facilitate an operation of sampling, reduce cost, and the like, a single-use system using a flexible vessel made of plastic has been currently developed and put to practical use, in place of a conventional stainless steel or glass culture vessel.
In a culture using the single-use system, a sterile bag is previously sterilized with ethylene oxide gas or gamma radiation. A sterilized liquid medium and a cell to be cultured are then put into the sterile bag. Also in the single-use system as described above, a very small quantity of the cell culture solution in the sterile bag is sampled under the bacteria-free condition at fixed time intervals; and an externally-provided analyzer measures the pH, dissolved oxygen concentration, carbon dioxide concentration, a cell concentration, and the like of the cell culture solution, so as to monitor a state of cultured cells in the culture vessel.
Such an operation of monitoring using the single-use system involves a risk of contamination and a loss of the cell culture solution. If continuous monitoring is necessary, such an operation is thus not always appropriate. Hence, there is a need for developing measurement technology in which the above-mentioned various measurement sensors are suitably adapted to the single-use system. For example, when continuous monitoring is required, a chip type fluorescent sensor is used in which: a fluorescent chip having fluorescent dye whose color development changes depending on the pH, dissolved oxygen concentration, or carbon dioxide concentration of the cell culture solution, is placed in a sterile bag; and fluorescence is detected and measured using an optical means from outside of the sterile bag.
Nonetheless, the turbidity sensor which is mainly used for measuring a cell concentration based on turbidity is of the aforementioned type used by being inserted in a culture vessel. Further, almost all types of the turbidity sensor are suited to being used with a culture vessel made of stainless steel or glass. There is thus need for developing a turbidity sensor usable in the single-use system and also developing a cell culture apparatus making use of the turbidity sensor. The inventions directed to such technologies are described in, for example, Patent Documents 1 to 3.
Japanese Laid-Open Patent Application, Publication No. H03-10677 (to be referred to as Patent Document 1 hereinafter) discloses a culture bag into which a cell and a culture medium are put to be cultured therein. The culture bag includes: a bag main body for receiving the cell and the culture medium; and an optical sensor which is placed in the bag main body and optically detects a concentration of the cells in the bag main body.
Japanese Laid-Open Patent Application, Publication No. 2012-189386 (to be referred to as Patent Document 2 hereinafter) discloses a component analyzer which includes: an adjustment device that adjusts a thickness of a flexible container as a target to be measured; and a measuring device that has a function of emitting near infrared light and a function of receiving the emitted near infrared light. The adjustment device includes a first adjustment part and a second adjustment part which adjust a thickness of an outer portion of the flexible container, the outer portion being a portion nearer to an edge of the flexible container with respect to a central portion thereof. The thickness of the outer portion of the flexible container is adjusted in such a manner that a first measuring surface contacts an outer portion of a first surface of the flexible container and a second measuring surface of the second adjustment part contacts an outer portion of a second surface of the flexible container. The measuring device has a function of emitting near infrared light from the first measuring surface toward the second measuring surface and a function of receiving the near infrared light at the second measuring surface.
International Publication No. WO 2012/127560 pamphlet (to be referred to as Patent Document 3 hereinafter) discloses a turbidity measurement device for measuring turbidity of a culture solution from outside a culture tank. The turbidity measurement device includes: a transparent part provided in a tank wall of a culture tank; a reflecting mirror disposed in a culture solution in the culture tank; a light emitter for irradiating light to the reflecting mirror via the transparent part, the light emitter being disposed outside the culture tank; and a detecting light receiver for receiving light from a direction of the reflecting mirror via the transparent part, the detecting light receiver being disposed outside the culture tank. The device described in Patent Document 3 measures turbidity by: disposing the reflecting mirror in the culture tank; irradiating light from the light emitter disposed outside the culture tank to the reflecting mirror; and detecting the reflected light having been reflected on the reflecting mirror, by the light receiver.